cis Golgi marker GM130 in control cells, while virtually below detection level in shRNA-a cells (Fig. 2B). To study the effect of DS-epi1 down regulation on DS structure, cells were labeled with [35S]-sulfate and PGs derived either from the cell layer or released into the medium were fractionated by size exclusion chromatography. Isolated [35S]-sulfate labeled CS/DS chains were specifically degraded at IdoA residues using chondroitinase B, and size fractionated (Fig. 2C). Calculation based on the degradation pattern demonstrated that the IdoA content was 7 % to 15 % in the control CS/DS-PGs, and was approx. 80 % reduced upon DS-epi1 silencing (Table I). In accordance with these results, IdoA-containing epitopes at the cell surface, measured by an anti-DS antibody, were reduced by 64 % and 47 % upon DS-epi1 downregulation by shRNA-a and shRNA-b, respectively (Fig. 2D). These data were corroborated by visualization of cell surface IdoA using an anti-DS antibody, which was substantially reduced IdoA staining was seen in DS-epi1 shRNA-a cells as compared to control cells (Fig. 2E). The remaining IdoA upon DS-epi1 downregulation could be formed by the action of DS-epi2, the mRNA expression of which increased 5-fold in DS-epi1-downregulated cells (Fig 2G), while, as expected, DS-epi1
